Sp1-Mediated Transcriptional Control of Fibroblast Growth Factor Receptor 4 in Sarcomas of Skeletal Muscle Lineage Shun Jiang Yu, 1 Lei Zheng, 1 Marc Ladanyi, 2 Sylvia L. Asa, 3 and Shereen Ezzat 1 1 Department of Medicine, Mount Sinai Hospital and University of Toronto, The Freeman Centre for Endocrine Oncology and The Ontario Cancer Institute, Toronto, Ontario, Canada; 2 Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York; and 3 Department of Pathology University Health Network and University of Toronto, The Freeman Centre for Endocrine Oncology and The Ontario Cancer Institute, Toronto, Ontario, Canada ABSTRACT Fibroblast growth factor receptors (FGFRs) have been implicated in a multitude of differentiating and proliferative actions. FGFR4 is expressed mainly in lung, kidney, pan- creas, spleen, and developing muscle. FGFR4 was found to be overexpressed in some human malignancies, where it has been implicated in their pathogenesis. Recently, FGFR4 was found to be overexpressed in pediatric rhabdomyosarcomas, based on cDNA microarray analysis. Using Northern blot- ting, reverse transcription-polymerase chain reaction, and Western blotting, we classified four human rhabdomyosar- coma-derived cell lines based on their relative expression of FGFR4. We defined a 214 bp (115/99) promoter that functioned as a minimal promoter and examined cis-DNA elements implicated in the control of expression of the FGFR4 gene in these cells. Overlapping 40- to 50-bp frag- ments of the minimal promoter were examined by electro- phoretic mobility shift assay using nuclear extracts from cell lines with high (HS729-1015) or low (HS729-1016) FGFR4 expression. Fragment C (65/26) formed specific com- plexes with nuclear extracts from both cell lines. Fragment B(95/56), however, formed distinct complexes mainly with the high FGFR4-expressing HS729-1015 cells. Both fragments yielded complexes that were competed by an Sp oligonucleotide and supershifted by Sp1 and by Sp3 anti- bodies. Transfection of Sp1 but not Sp3 efficiently activated FGFR4 promoter activity, an effect that was significantly more pronounced in the HS729-1015 cell line than in the low FGFR4-expressing HS729-1016 cell line. Deletion of each of the two Sp-binding sites in fragments B and C resulted in loss of promoter activity. In particular, deletion of the 5 Sp-binding site in fragment B was associated with the great- est loss of activity. Sp1 protein expression correlated with FGFR4 expression in cell lines and primary human rhab- domyosarcomas. Furthermore, transfection of Sp1 and methylation inhibition was effective in inducing the endog- enous FGFR4 gene in HS729-1015 cells. Our findings point to Sp1 as an important contributor to FGFR4 transcrip- tional control and elucidate a potential mechanism for the heterogenous expression of FGFR4 in neoplasms derived from the same cell lineage. INTRODUCTION Myogenic cells proliferate as mononucleated myoblasts before differentiating into postmitotic multinucleated skeletal muscle fibers. This process is governed by signal transduction cascades heavily orchestrated by growth factors. Of these, mem- bers of the fibroblast growth factor (FGF) and FGF receptor (FGFR) family have been implicated in sustaining myoblast proliferation and possibly delaying their differentiation (1). FGF-1 and FGF-2 possess well-documented mitogenic activity on skeletal muscle cells; both can activate FGFR1 in prolifer- ating myoblasts (2). Human skeletal muscle-derived malignan- cies are referred to as rhabdomyosarcoma and are well known to express the FGF-2 gene and protein (3). Moreover, FGF-2 stimulation of myoblasts induces SHP-2 complex formation with fibroblast growth factor receptor substrate (FRS2) and induces Erk activity and Elk-1 transactivation. Overexpression of SHP-2 potentiates the suppressive effects of FGF-2 on muscle-specific gene expression and myogenesis (4), further highlighting the importance of FGFR signaling in myogenic differentiation. FGF signaling is mediated through four dedicated FGFRs. There are currently four known mammalian FGFR genes en- coding a complex family of transmembrane receptor tyrosine kinases (5). Each prototypic receptor is composed of three immunoglobulin-like extracellular domains, a single transmem- brane domain, a split tyrosine kinase, and a COOH-terminal tail with multiple autophosphorylation sites (5). Multiple cell-bound or secreted forms of all FGFRs result from alternative transcrip- tion initiation, alternative splicing, exon switching, or variable polyadenylation. Recent evidence has emphasized the importance of FGFR4 in skeletal muscle. There is marked loss of skeletal muscle development in chick embryos resulting from loss of FGFR4 signaling (6). FGFR4 is also expressed in lung, kidney, adrenal gland, pancreas, and spleen (7, 8). The role of FGFR4 in carcinogenesis is poorly understood. FGFR4 mediates mem- brane ruffling in breast carcinoma cells (9), where it is overex- pressed (10), and it has been shown to modulate erythroid cell proliferation (11). A tumor-derived FGFR4 (ptd-FGFR4) iso- form recapitulates pituitary tumorigenesis in transgenic mice Received 2/4/04; revised 5/2/04; accepted 5/12/04. Grant support: The Cancer Research Society, Canadian Institutes of Health Research (grant MT-14404), and Toronto Medical Laboratories. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Shereen Ezzat, University of Toronto-Mount Sinai Hospital, 600 University Avenue #437, Toronto, Ontario, M5G 1X5 Canada. Phone: 416-586-8505; Fax: 416-586-8834; E-mail: sezzat@mtsinai.on.ca. ©2004 American Association for Cancer Research. 6750 Vol. 10, 6750 – 6758, October 1, 2004 Clinical Cancer Research Research. on June 26, 2021. © 2004 American Association for Cancer clincancerres.aacrjournals.org Downloaded from